Greening contested the constituency of Ealing, Acton & Shepherd's Bush in 2001, finishing second with a reduced share of the vote for the Conservatives. She won the seat of Putney in the 2005 General Election on 5 May 2005. Greening won 15,497 votes (42.4% of the vote) giving her a majority of 1,766 (4.8%). She unseated Tony Colman, who had held the seat for Labour since defeating David Mellor in 1997. As the first Conservative elected on the evening of the election, her victory was the first real sign that the Conservative Party was to reduce the Labour government's majority and begin to recover from the landslide defeats of the 1997 and 2001 General Elections. Michael Howard, who had visited Putney to give a speech on his first day as Conservative Leader, returned there on the morning after the Election to congratulate Putney Conservatives and give the speech in which he announced his intention to step down.

Greening was appointed a vice-chairman (with responsibility for Youth) of the Conservative Party on 15 December 2005, having earlier that year been appointed a member of the Work and Pensions Committee. In July 2007 following a Shadow Ministerial reshuffle, she was promoted to be a junior Shadow Minister for the Treasury. In January 2009 following a further Shadow Ministerial reshuffle, Greening was promoted to Shadow Minister for London, within the Communities and Local Government Team with responsibility for Local Government Finance. Within this brief, she focussed on transport and local community benefits. In March 2010 she was put in charge of co-ordinating the Conservative campaign for the 2010 General Election in London.[5] She became Economic Secretary to the Treasury.

In October 2011 she became Secretary of State for Transport and was appointed a Privy Councillor.[6]

Greening represents the London constituency of Putney and had always campaigned against a third runway at Heathrow. In the run up to the 2012 Cabinet reshuffle Greening said it would be difficult to serve in a Cabinet which was in favour of a third runway.[7]

In the event Greening was reshuffled on 4 September 2012. The move was attacked by Boris Johnson.[8]

Greening failed to vote in a critical parliamentary division about military action in Syria. Although in the House of Commons at the time, she was reportedly chatting to another minister in a room behind the chamber and failed to notice that the division bell had sounded.[10]

She was the youngest female Conservative MP in the House of Commons[11] until Chloe Smith (who coincidentally succeeded her at HM Treasury) was elected to Parliament on 12 October 2009.

During the 2009 expenses scandal, Greening was ranked as the 9th best value for money MP in research carried out by the free-market think tank, the Adam Smith Institute, and of all her expenses claims she was the 599th lowest out of the 645 MPs.